Project description:Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset syndrome characterized by progressive degeneration of particular muscles. OPMD is caused by short GCG repeat expansions within the gene encoding the nuclear poly(A)-binding protein 1 (PABPN1) that extend an N-terminal polyalanine tract in the protein. Mutant PABPN1 aggregates as nuclear inclusions in OMPD patient muscles. We have created a Drosophila model of OPMD that recapitulates the features of the human disorder: progressive muscle degeneration, with muscle defects proportional to the number of alanines in the tract, and formation of PABPN1 nuclear inclusions. Wild-type human PABPN1 contains a stretch of 10 alanines following the initial methionine, which is expanded to 12–17 alanines in OPMD patients. In Drosophila, the PABPN1 homolog is the poly(A)-binding protein 2 (PABP2), which has the same function as PABPN1 in nuclear polyadenylation but lacks a polyalanine tract at the N-terminus. We used the UAS/Gal4 system to express mammalian PABPN1 in Drosophila. An alanine-expanded PABPN1 cDNA (encoding the 17 alanine tract) was cloned downstream of UAS sequences (UAS-PABPN1). Transgenic lines containing this construct were crossed to a Mhc-Gal4 driver, leading to muscle-specific expression. To gain insight into the molecular and physiological defects in OPMD we performed a transcriptomic analysis in OPMD fly muscles. Using microarrays, thorax gene expression was compared between control flies (Mhc-Gal4/+) and flies expressing PABPN1-17ala in thoracic muscles (UAS-PABPN1-17ala/+; Mhc-Gal4/+), at three time points (days 2, 6 and 11). Transcriptome of thorax RNA samples from control (Mhc-Gal4/+) flies and flies expressing PABPN1-17ala (UAS-PABPN1-17ala/+; Mhc-Gal4/+)
Project description:Transcriptional profiling of Indy long lived flies and controls over the course of their entire lifespan. Mutations in the Indy gene extend life span in Drosophila melanogaster. This study investigates the changes in gene expression over time in Indy206 flies heterozygous over Canton-S (Indy206/CS) and compares them to genetically matched heterozygous controls (2216/CS). Samples from both fly strains were collected at age: 5, 10, 20, 30, 40, 50, 70 and 80. mRNA samples were collected from the head and thorax of Indy206 and genetically matched control male adult flies at day 5, and every 10 days from day 10 to day 80 (day 60 excluded) and hybridized to two-color microarrays
Project description:Protein expression profiles in the whole tissue lysate extracted from the thorax of O1 and O3 long-lived flies with the control B3 strain flies and analyzed by tandem mass tag (TMT)–based mass spectrometry.
Project description:Dietary restriction (DR) is one of the most studied interventions known to extend life span. The robustness of its effect across species suggests the existence of conserved mechanisms to reduce mortality rates and increase longevity. However, because DR elicits a large number of physiological changes, many of which are unrelated to the longevity response, it has been difficult to identify these specific mechanisms. Whole-genome gene expression studies have typically reported several hundreds to thousands of differentially expressed genes in response to DR. The fruit fly Drosophila melanogaster shows a remarkable response to a change in diet: after a switch to DR, food mortality rates drop within 2-4 days to the same level as cohorts continuously on DR. Based on this observation, we utilized a novel experimental design to enrich for genes directly associated with the longevity response. By profiling gene expression in a cohort of fruit flies that were switched from normal food to DR we were able to partition genes in several classes with distinct patterns of expression. Canton-S flies were kept at 25˚C in a temperature-controlled incubator at 50% humidity with a 12 hour on/off light cycle. The flies were sorted into vials at a density of 25 males and 25 females per vial, randomly divided into treatment groups and passed every day on either Control Food (CF, 150 g/L sucrose, 150 g/L autolysed yeast, and 20 g/L agar, all w/v) or Restricted Food (RF, 50 g/L sucrose, 50 g/L autolysed yeast, and 20 g/L agar, all w/v) and the number of dead flies recorded. On day 40, half of the flies on Control Food were switched to the Restricted Food. During the course of the experiment, age-specific instantaneous mortality rate was analyzed and the separation of mortality rate between the food conditions was verified before the switch. Flies were sorted under light CO2 and collected at fixed time intervals during (0 = time of switch), and after (2, 4, 6, 8, 12, 18, 24, 32, 40, 48, 56, 72 hours after) the switch time point via snap freezing in liquid nitrogen and were stored at −80°C. Heads and thorax of female flies were collected for microarray experiments.
Project description:Reduced PABPN1 levels cause aging-associated muscle wasting. PABPN1 is a multi-functional regulator of mRNA processing. To elucidate the molecular mechanisms causing PABPN1-mediated muscle wasting, we compared the transcriptome to the proteome in mouse muscles expressing shRNA to PABPN1 (shPab). We found greater variations in the proteome as compared to mRNA expression profiles. Protein accumulation in the shPab proteome was concomitant with reduced proteasomal activity. Notably, protein acetylation appeared to be enriched in shPab versus control proteomes (63%). An acetylome study in shPab muscles revealed prominent peptide deacetylation associated with elevated sirtuin-1 (SIRT1) deacetylase. We show that SIRT1 mRNA levels are controlled by PABPN1 via an alternative polyadenylation site utilization. SIRT1 inhibition reversed PABPN1 activity and muscle cell function. Moreover, deacetylation inhibition increased PABPN1 levels and reversed muscle wasting. We suggest that perturbation of a multifactorial regulatory loop involving PABPN1 and SIRT1 plays an imperative role in aging-associated muscle wasting.